Ubiquitylation is a post-translational modification best known for its role in protein degradation. While ubiquitin is absent from mitochondria, mitochondrial outer membrane (MOM) proteins are exposed to cytosol and are thus accessible to the ubiquitylation machinery. E3 Ub ligases confer specificity to ubiquitylation. We recently annotated the E3s in the human genome and generated collections of arrayed cDNA and RNAi representing nearly the entire family, for functional genomic approaches. In a screen using these collections, we uncovered MULAN, a novel E3 that localizes to mitochondria and activates NF-(B. Objective/Hypothesis: The goals are to elucidate the function and regulation of MULAN in NF-(B signaling. The hypotheses being tested are that MULAN acts as a mitochondrial regulator of NF-(B activation, apparently acting upstream of IKK and p65, that MULAN functions by interacting with and activating a downstream effector, and that MULAN's E3 activity can play a negative role in signaling by mediating the protein's auto- ubiquitylation and degradation. The Specific Aims are to further characterize the activation of NF-(B by MULAN and investigate mechanisms involved, to identify MULAN-interacting proteins involved in signaling to NF-(B, and to investigate how MULAN's E3 activity regulates the protein's expression. Study design: Biochemical and genetic approaches will be used to identify MULAN's downstream effectors. cDNA expression and siRNA-mediated loss-of-function analyses will test their relevance for NF-(B activation and will order the proteins in a pathway based on epistasis. Their role and mechanisms in MULAN-mediated NF-(B activation will be investigated biochemically. In vitro and in vivo assays for E3 activity will examine MULAN's regulation. Rapid biochemical changes elicited in response to the conditional expression of MULAN will be determined. Lastly, biochemical and siRNA analyses will investigate the pathway leading to MULAN's degradation from the MOM. Cancer relevance: The relevance of the NF-(B pathway for cancer is underscored by the finding of mutations in some of its pathway components in melanoma, colon and liver cancers as well as by the pathway's critical role in inflammation. Small molecules targeting the pathway are in clinical development. The results of the proposed studies should ultimately translate into the identification of new targets for drug discovery. PUBLIC HEALTH RELEVANCE Several biochemical pathways used for communication within cells have been implicated in cancer through epidemiology and molecular genetic studies, such as the pathway leading to a protein regulator of gene activity known as NF-(B. We discovered a gene whose product controls the activation of NF-(B by a novel mechanism, and its role in cancer is also suggested by apparently elevated levels in melanoma and glioblastoma cells. Characterization of this protein and how it connects to NF-(B will significantly enhance our basic understanding of the NF-(B pathway, and should provide new targets and opportunities for drug discovery in Oncology, as well as other indications such as inflammatory diseases.